Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

Abstract

Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs) are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP) and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with N-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or d-galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity.

Increased ethanol‐inducible cytochrome P450‐2E1 and cytochrome P450 isoforms in exosomes of alcohol‐exposed rodents and patients with alcoholism through oxidative and endoplasmic reticulum stress

“…EVs were isolated from the plasma of patients with alcoholism and control individuals in order to evaluate the increased secretion of P450 proteins, including CYP2E1, in EVs by alcohol exposure . Compared to the age‐matched controls (n = 9, 17.2 ± 3.3), serum aspartate aminotransferase levels in patients with alcoholism (n = 14, 69.9 ± 33.4) were significantly increased, indicating alcohol‐induced liver damage.…”

“…The inset figures represent transmission electron microscopy images. (B) EV size was determined by a Nanoparticle Tracking analysis (Nanosight) . (C) Total number of EVs in the sera of healthy controls or patients with alcoholism was measured by Nanosight.…”

“…This study was approved by the Human Subjects Institutional Review Board of the Johns Hopkins University Medical Institutions, Baltimore, MD. The clinical characteristics of the healthy controls and patients with alcoholism are the same as described . Individual EV proteins were prepared from the serum samples of the study subjects as described …”

Ascorbic Acid Attenuates Acute Ethanol-Induced Liver Injury in SMP30 Knockout Mice

“…Previous studies have reported that a single dose of ethanol at 6 g/kg administered by gavage successfully [17] causes liver histopathologies, such as microvesicular steatosis and necrosis in hepatocytes, which are characterized by cell enlargement and nuclear dissolution [19]. In the present study, the same ethanol dose (6 g/kg) was administered to the mice once a day for three days.…”

“…Mice in the ethanol-treated groups were exposed to an acute dose of 6 g ethanol/kg body weight administered by gavage once a day for three days (0, 24, and 48 h), whereas control mice received an equal volume of water by oral administration. [17] All animals were sacrificed at 12 h after the third ethanol administration (Table 1).…”